1. Field of the Invention
This invention is in the field of particle detectors. More particularly, it relates to a detector for detecting the electrokinetic passage of minute quantities of particles past a reference point in a microcolumn.
2. Description of Background Materials
Several analytical methodologies have been developed in which a fluid sample is driven through a narrow bore microcolumn so as to separate and/or isolate the various particles and species contained within the fluid sample on the basis of size, shape, charge, viscosity, mobility, polarity, solvent-solute inter-reaction, or the like. Two of these methodologies based on packed columns are isotachophoresis and high performance liquid chromatography. Another process of growing importance is electrokinetic separation (also commonly referred to as open tubular electrophoresis separation or capillary zone electrophoresis separation).
With electrokinetic separation, as with any separation process, it is necessary to have a means to detect the passage of particles or species through the column and/or the arrival of particles or species at a set location in the column after the separation has taken place. A wide range of detectors are known based on any one of many changes in properties which may occur as the particles or species pass the detection zone. These can include, for example, a change in optical properties (e.g. U.V., visible or I.R. based detectors as well as refractive index detectors), a change in electrical properties (e.g. conductance or resistance-based detectors), or a change arising from an electrochemical reaction (e.g. amperometric, coulometric, or potentiometric detectors).
The present invention relates to an improved electrical detector for use with microcolumn electrokinetic separation systems. Electrical detectors can be distinguished from electrochemical detectors. Electrochemical detectors involve electric effects due to chemical changes which occur when a particle or species enters the detection zone. Electrical detectors respond to changes in the conductance of current or changes of resistance which result when particles or species enter the detection zone. With electrical detectors no chemical reaction is necessarily associated or required.
With any microcolumn electrokinetic separation process and any of these detector methodologies, there is an interest in increasing the sensitivity. This can lead to smaller sample sizes being used or to detection of smaller trace components in the samples.
A number of workers have proposed a variety of small volume high sensitivity detectors for use on various types of columns. For example, Jorgenson, et al. (Knecht, L. A., Guthrie, E. J., and Jorgenson, J. W. Anal. Chem., 1984, 56, 479-82; St. Claire, R. L., III, and Jorgenson, J. W. J. Chromatogr. Sci., 1985, 23, 186-91) built an on-column electrochemical detector for a 15 micron internal diameter open tubular column. In this system the working electrode was a 5 micron or 9 micron carbon fiber that was inserted with a micropositioner into the end of the capillary. Adler, et al. (Adler, J. F., Fielden, P. R., and Clark, A. J. Anal. Chem., 1984, 56, 985-988) described a combination conductivity/permittivity detector. This detector provided simultaneous measurements of these two properties within a single cell. The detector was applied to ion chromatography systems. A detection limit of 40 ppb of chloride was reported. This system employed a pair of conical electrodes as its detection cell. Doury-Berthod, et al. (Doury-Berthod, M., Giampoli, P., Pitsch, H., Sella, C., and Poitrenaud, C. Anal. Chem., 1985, 57, 2257-2263) presented a theoretical description of dual column chromatography with conductivity detection. Their analytical response appears to be the sum of the conductometric contribution of the solute and the eluant. Kaniansky, et al. (Kaniansky, D., Koval, M., and Stankoviansky, S. J. Chromatogr., 1983, 267, 67-73) use 0.01 mm Pt-Ir alloy wires as electrodes in isotachophoresis. The capillaries were as small as 0.1 mm and made of fluoropolymers (PTFE, FEP). The wires were heated and pushed through the walls of the capillary. T. Tsuda has also described the use of a commercial conductivity detector external to the capillary for analyzing small positively charged metal ions (Suzuken Memorial Foundation 3, 33 (1984)).
Several papers by Mikkers, et al. (Mikkers, F. E. P., Everaerts, F. M., and Peek, J. A. F., J. Chromatogr., 1979, 168, 317-332; Mikkers, F. E. P., Everaerts, F. M., and Verheggen, Th. P. E. M. J. Chromatogr. 1979, 169, 1-10; Mikkers, F. E. P., Everaerts, F. M., and Verheggen, Th. P. E. M. J. Chromatogr. 1979, 169, 11-20) all referenced conductivity detectors for isotachophoresis in capillary electrophoresis based on the work of Everaerts and colleagues (Everaerts, F. M., and Verheggen, Th. P. E. M. J. Chromatogr. 1972, 73, 193-210; Everaerts, F. M., and Verheggen, Th. P. E. M. J. Chromatogr., 1974, 91, 837-851; Everaerts, F. M., and Rommers, P. J. J. Chromatogr., 1974, 91, 809-818; Everaerts, F. M., Geurts, M., Mikkers, F. E. P., and Verheggen, Th. P. E. M. J. Chromatogr., 1976, 119, 129-155; Kaniansky, D., and Everaerts, F. M. J. Chromatogr., 1978, 148, 441-446; Everaerts, F. M., Beckers, J. L., and Verheggen, Th. P. E. M. "Isotachophoresis: Theory, Instrumentation and Applications", 1976, Elsevier, New York, p. 136). The Everaerts' device consists of glass or PTFE capillary tubing with an inside diameter of 0.4 to 0.6 mm and an outside diameter of 0.7-1 mm. Two blocks of capillary tubing have one of each of their ends fixed in a block so that the two blocks can be clamped together. Before clamping, two disks of insulating material 0.005 mm thick have platinum sputtered on both sides and separated from one another by a disk of plain insulating material are drilled to form a hole in their centers which matches the i.d. of the capillary. These disks are placed between the two blocks which are then clamped so that no leakage occurs.
Bocek et al. (Foret, F., Deml., M., Kahle, V., and Bocek, p., Electrophoresis 1986, 7, 430-432) developed a conductivity cell for capillary zone electrophoresis. They used capillaries of about 0.3 mm i.d. Platinum electrodes were molded into a polyester block containing a channel with a circular cross-section equal to the inside diameter of the capillary. Larger openings in the ends of the block made a tight fit with pieces of capillary inserted. In both the Everaerts and Bocek cells, it is important to note that the electrodes are outside the capillary and that the inside surface of the glass or PTFE tubing is not continuous. These discontinuities in the tubing surface can be of particular concern in column electrophoresis or isotachophoresis systems. In these systems substantial voltages are connected across the column. Any increase in column cross-sectional area or any discontinuity in the column surface can lead to disturbances in the flow. This can reduce the accuracy and reproducibility of results. This problem becomes especially acute as one attempts to use smaller and smaller cross-section columns and higher and higher driving voltages. Another problem arises when the cross-section increases at the detector because of dead or void volume which can diminish the sensitivity of the detector. For these reasons, we have found that it is most advantageous in these settings to present as continuous an internal surface as possible with no discontinuities or cross-section increase.